The invention relates to a method of inhibiting cellular and humoral responses to immunostimulatory DNA with compositions including 4-aminoquinolines, 9-aminoacridines and derivatives thereof.
Bacterial DNA is increasingly recognized as a powerful modulator of immunity (Krieg, 1997. Trends in Microbiol. 4:73-6), stimulating the polyclonal proliferation of B-cells and the production of cytokines by monocytes and other cells (Ballas et al., 1996. J. Immunol. 157:1840-5). This activity is attributed to unmethylated CpG sequences in bacterial DNA, which are uncommon in vertebrate DNA (Krieg et al., 1995. Nature 374:546-9). Single stranded oligodeoxynucleotides which have the motif PuPuCGPyPy (CpG-ODN) mimic many of the actions of bacterial DNA (Krieg et al., 1996. Antisense and Nucleic Acid Drug Devel. 6:133-9), and powerfully inhibit the induction of apoptosis in mouse WEHI 231 B-cells by anti-surface IgM (Yi et al., 1996. J. Immunol. 157:4918-25; Macfarlane et al., 1997. Immunology 91:586). This system is a convenient and reproducible model to study responses to immunostimulatory DNA.
Bacterial DNA immobilized on beads does not stimulate immune responses, suggesting that internalization of the DNA is required for activity. DNA and oligodeoxynucleotides are endocytosed into acidic vesicles, and are then transported to the cytoplasm and nucleus of cells.
Chloroquine, hydroxychloroquine and quinacrine are known to induce remissions of systemic lupus erythematosus and rheumatoid arthritis by an unknown mechanism. These drugs bind to DNA by intercalation. They are weak bases and partition into acidic vesicles. At high concentrations, chloroquine can collapse the pH gradient and disrupt the action of endosomal hydrolytic enzymes and the trafficking of receptors.
The invention is based on the discovery that quinacrine, chloroquine and selected 9-aminoacridine and 4-aminoquinoline compounds unexpectedly block the action of immunostimulatory DNA in cells at concentrations much below those needed for the other immunomodulatory effects of these compounds in vitro. The 9-aminoacridine and 4-aminoquinoline compounds utilized in the methods of the invention inhibit the anti-apoptotic effect of CpG-ODN and the CpG-ODN-induced secretion of IL-6. On the other hand, these compounds do not influence the anti-apoptotic effects of other agents, for example, lipopolysaccharides. The effects of the compounds are highly specific to immunostimulatory DNA. These agents are useful for inducing remissions of autoimmune disorders such as rheumatoid arthritis and systemic lupus erythematosus, leading to generally useful remittive and anti-inflammatory agents.
The methods of the invention are useful for inhibiting immunostimulatory DNA-associated responses in a subject, and involve the administration of a composition to a subject exhibiting responses initiated or exacerbated by immunostimulatory DNA. The compositions include 4-aminoquinolines, 9-aminoacridines and derivatives thereof. These compounds may be linked to each other by linkers. The responses initiated or exacerbated by immunostimulatory DNA include those which result in the initiation or exacerbation of various disease states. The disease states include septic shock, inflammatory bowel diseases, asthma, sinusitis, various autoimmune disorders such as hemolytic anemia, and others. The responses to be inhibited include the production of immunomodulatory proteins such as cytokines, interleukins, interferons, cell growth factors and chemokines. One such protein is IL-6. Other responses to be inhibited by the methods of the invention are high levels of erythrocyte sedimentation, and high levels of immunoglobulin.
The invention also features a method of screening compounds useful for inhibiting immunostimulatory DNA-associated responses. This method involves contacting a cell with immunostimulatory DNA, thereby inducing a measurable immune response, and a test compound. Any inhibition of the immune response is detected. The cells can be hybridoma plasma cells, including 7TD1.
A xe2x80x9cnucleic acidxe2x80x9d or xe2x80x9cDNAxe2x80x9d means multiple nucleotides (i.e., molecules comprising a sugar (e.g., ribose or deoxyribose)) linked to a phosphate group and to an exchangeable organic base, which is either a substituted pyrimidine (e.g., cytosine (C), thymine (T) or uracil (U)) or a substituted purine (e.g., adenine (A) or guanine (G)). As used herein, the term refers to ribonucleotides as well as oligodeoxynucleotides. The term also includes polynucleosides (i.e., a polynucleotide minus the phosphate) and any other organic base-containing polymer. Nucleic acid molecules can be obtained from existing nucleic acid sources (e.g., genomic or cDNA), but are preferably synthetic (e.g., produced by oligonucleotide synthesis). A xe2x80x9cstabilized nucleic acid moleculexe2x80x9d or xe2x80x9cnuclease-resistant nucleic acid moleculexe2x80x9d means a nucleic acid molecule that is relatively resistant to in vivo degradation (e.g., via an exo- or endo-nuclease). Stabilization can be a function of length or secondary structure. Unmethylated CpG-containing nucleic acid molecules that are tens to hundreds of kilobases long are relatively resistant to in vivo degradation. For shorter immunostimulatory nucleic acid molecules, secondary structure can stabilize and increase their effect. For example, if the 3xe2x80x2 end of a nucleic acid molecule is self-complementary to an upstream region, so that it can fold back and form a stem loop structure, then the nucleic acid molecule becomes stabilized and therefore exhibits more activity.
Preferred stabilized or nuclease-resistant nucleic acid molecules referred to in the instant invention have a modified backbone. For use in immune stimulation, especially preferred stabilized nucleic acid molecules are phosphorothioate-modified nucleic acid molecules (i.e., at least one of the phosphate oxygens of the nucleic acid molecule is replaced by sulfur). Preferably the phosphate modification occurs at or near the 5xe2x80x2 and/or 3xe2x80x2 end of the nucleic acid molecule. In addition to stabilizing nucleic acid molecules, phosphorothioate-modified nucleic acid molecules (including phosphorodithioate-modified) can increase the extent of immune stimulation of the nucleic acid molecule, which contains an unmethylated CpG dinucleotide.
International Patent Application Number WO 95/26204 entitled xe2x80x9cImmune Stimulation by Phosphorothioate Oligonucleotide Analogsxe2x80x9d also reports on the non-specific immunostimulatory effect of phosphorothioate modified oligonucleotides. International Patent Application WO 96/02555 discloses the ability of unmethylated CpG-containing oligonucleotides to activate lymphocytes. Unmethylated CpG-containing nucleic acid molecules having a phosphorothioate backbone have been found to preferentially activate B-cell activity, while unmethylated CpG-containing nucleic acid molecules having a phosphodiester backbone have been found to preferentially activate monocytic (macrophages, dendritic cells and monocytes) and NK (natural killer) cells. Other stabilized nucleic acid molecules include non-ionic DNA analogs, such as alkyl- and aryl-phosphonates (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphotriesters, in which the charged oxygen moiety is alkylated. Nucleic acid molecules that contain a diol, such as tetraethyleneglycol or hexaethyleneglycol, at either or both termini have also been shown substantially resistant to nuclease degradation.
As used herein, the term xe2x80x9cimmunostimulatory DNAxe2x80x9d refers to bacterial DNA, viral DNA, other DNA, synthetic double- or single-stranded DNA, DNA synthesized with a nuclease-resistant backbone or at least a partially nuclease resistant backbone, (such as a phosphorothioate-backboned DNA) which stimulates (e.g., has a mitogenic effect on, or induces or increases cytokine expression by vertebrate lymphocytes. Such immunostimulatory DNA includes that which contains an unmethylated cytosine, guanine (CpG) dinucleotide sequence. A xe2x80x9cCpGxe2x80x9d or xe2x80x9cCpG motifxe2x80x9d refers to a nucleic acid having cytosine followed by a guanine linked by link containing phosphorus. The term xe2x80x9cmethylated CpGxe2x80x9d refers to the methylation of the cytosine on the pyrimidine ring, usually occurring at the 5xe2x80x2-position of the pyrimidine ring. The term xe2x80x9cunmethylated CpGxe2x80x9d refers to the absence of methylation of the cytosine on the pyrimidine ring. Methylation, partial removal, or removal of an unmethylated CpG motif in an oligonucleotide of the invention is believed to reduce its effect. Methylation or removal of all unmethylated CpG motifs in an oligonucleotide substantially reduces its effect. The effect of methylation or removal of a CpG motif is xe2x80x9csubstantialxe2x80x9d if the effect is similar to that of an oligonucleotide that does not contain a CpG motif.
As used herein, the term xe2x80x9cairway obstructionxe2x80x9d includes respiratory ailments such as asthma, sinusitis, as well as airway obstructions caused by an infection, or exacerbated by an infection.
The terms xe2x80x9clower alkylxe2x80x9d and xe2x80x9clower alkoxyxe2x80x9d refer to alkyl- and alkoxy groups comprising up to 7 carbon atoms. The terms include straight and branched chain groups. The term xe2x80x9calicylicxe2x80x9d refers to a non-aromatic carbon chain system forming a ring, and having from 4 to 10 members. The term xe2x80x9cpolyalicyclicxe2x80x9d refers to a non-aromatic carbon chain system forming more than one ring, each ring having from 4 to 10 members. The term xe2x80x9caryl groupxe2x80x9d refers to aromatic hydrocarbons having from 6 to 26 carbons.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods and examples are illustrative only and not intended to be limiting.